CN115197847B - Probiotic freeze-dried tablet and preparation method thereof - Google Patents
Probiotic freeze-dried tablet and preparation method thereof Download PDFInfo
- Publication number
- CN115197847B CN115197847B CN202210950393.7A CN202210950393A CN115197847B CN 115197847 B CN115197847 B CN 115197847B CN 202210950393 A CN202210950393 A CN 202210950393A CN 115197847 B CN115197847 B CN 115197847B
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- freeze
- probiotic
- drying
- dried tablet
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Abstract
The invention belongs to the technical field of microbial preparation, and particularly relates to a probiotic freeze-dried tablet and a preparation method thereof. The probiotics freeze-dried tablet provided by the invention comprises the following components in parts by weight: 20-40 parts of a composite probiotic, 10-20 parts of galactooligosaccharide, 8-12 parts of oat beta-glucan, 10-20 parts of a freeze-drying protective agent, 5-8 parts of a gingko extract, 2-5 parts of a Chinese yam extract, 0.5-2 parts of vitamin C, 0.8-1.8 parts of vitamin E and 1.2-2.5 parts of stevioside. The probiotic freeze-dried tablet provided by the invention has excellent freezing resistance and storage stability, can more effectively resist the inactivation or death of probiotics caused by a freeze-drying process in the freeze-drying process, effectively improves the survival rate of live bacteria of the freeze-dried tablet, and can also improve the gastric acidity resistance of the probiotic freeze-dried tablet.
Description
Technical Field
The invention belongs to the technical field of microbial preparation, and particularly relates to a probiotic freeze-dried tablet and a preparation method thereof.
Background
Probiotics are active microorganisms which can produce beneficial effects on host health or physiological functions by promoting the ecological balance of various microbial flora in the intestinal tracts of the hosts, and can have definite health effects, improve and maintain the microecological balance and exert the probiotics effect. The probiotics have various health protection and disease defense functions for human bodies, and the metabolites of the probiotics can inhibit the growth and propagation of part of pathogenic bacteria by regulating the acid environment of the organisms, so that the probiotics are beneficial to the health of the human bodies. It can prevent diarrhea and other diseases by regulating intestinal probiotics flora; in addition, the effects of probiotics are widely accepted in the fields of foods, medicines and health products, but in practical application, the probiotics have the defects that: the probiotics are easy to be inactivated by the action of gastric acid, pepsin and the like after entering the human digestive tract, and the utilization rate is low; during production, processing, transportation and storage, the activity of probiotics is reduced due to adverse environmental effects, which is also a main reason that the probiotics cannot be widely applied to industrial production.
In order to enable long-term storage of the probiotic bacteria, it is preferably made dry. Currently, commonly used methods for preparing bacterial powders are air flow drying, freeze drying and spray drying. Freeze drying is one of the common means for protecting the number of living bacteria and biological activity in microbial fermentation agents at present, has wide application range and has good effect on various microorganisms; the survival rate of the viable bacteria of the fermentation powder is high, and the preservation time of the active ingredients is long. However, during the process, the probiotic cells die and the substances inside the cells are denatured during the freezing and drying processes, so that excessive cell deactivation is caused.
Chinese patent application CN112841657A discloses a multi-strain composite freeze-dried powder and a preparation process thereof, wherein the multi-strain composite freeze-dried powder comprises bacillus natto, monascus, probiotics, hawthorns and proteins, and the preparation process comprises the following steps: (1) preparation of bacillus natto; (2) Inoculating monascus strains to the solid culture medium for fermentation after sterilization treatment, and drying after secondary fermentation to obtain the monascus containing the bacillus natto; (3) Preparing a probiotic culture medium, (4) respectively screening bacillus natto, monascus, probiotics, hawthorn and protein, weighing the bacillus natto, the monascus, the probiotics, the hawthorn and the protein according to parts by weight, and grinding and crushing the bacillus natto, the monascus, the probiotics, the hawthorn and the protein; (5) obtaining the multi-strain composite freeze-dried powder. Chinese patent application CN 101926428A discloses a probiotic gingko pollen freeze-dried powder and a preparation method thereof, wherein the probiotic gingko pollen freeze-dried powder is a live bacteria freeze-dried powder prepared by taking wall-broken or non-wall-broken gingko pollen in the current year as a raw material, adding brown sugar in a certain proportion, fermenting by single strain or mixed fermentation of two probiotics of lactobacillus rhamnosus and lactobacillus acidophilus, and then freeze-drying. The number of the probiotics reaches 10 9 cfu/g or more, and contains a large amount of ginkgo pollen functional components and probiotics fermentation products. Although probiotics are added in the invention, the invention can theoretically play a role inThe host produces bacteriostats, regulates intestinal flora, enhances the efficacy of the body's immunity, but the added probiotics cause loss of activity due to the freeze-drying process exposing living cells to additional stress pressure. And the formation of intracellular ice crystals and recrystallization can cause cell membrane damage, so that part of living cells die, and meanwhile, the activity and the functional characteristics of the probiotics are influenced due to poor tolerance of the probiotics in the acidic environment of human bodies and poor storage stability of the probiotics in various microecologics.
Therefore, there is a need to develop a probiotic freeze-dried tablet that can effectively improve the survival rate of the probiotic after freeze-drying and improve the tolerance of the probiotic in environments such as gastric acid.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a probiotic freeze-dried tablet and a preparation method thereof. The probiotic freeze-dried tablet provided by the invention can effectively maintain the balance of microbial flora in intestinal tracts, promote the absorption of nutrient substances by human bodies, has a certain immunoregulatory effect on human bodies, has excellent freezing resistance and storage stability by reasonably compounding different types of freeze-drying protective agents in the preparation process, can effectively resist the inactivation or death of probiotics caused by a freeze-drying process in the freeze-drying process, effectively improves the survival rate of live bacteria of the freeze-dried tablet, can also improve the gastric acidity resistance of the probiotic freeze-dried tablet, enables the probiotics to be fixedly planted in the intestinal tracts, and better plays a role in health care.
The technical scheme of the invention is as follows:
the probiotics freeze-dried tablet comprises the following components in parts by weight:
20-40 parts of a composite probiotic, 10-20 parts of galactooligosaccharide, 8-12 parts of oat beta-glucan, 10-20 parts of a freeze-drying protective agent, 5-8 parts of a gingko extract, 2-5 parts of a Chinese yam extract, 0.5-2 parts of vitamin C, 0.8-1.8 parts of vitamin E and 1.2-2.5 parts of stevioside.
In the probiotic freeze-dried tablet prepared by the invention, the added ginkgo extract contains substances such as ginkgo total flavone, bilobalide and the like, and has the effects of dilating blood vessels, protecting vascular endothelial tissues, regulating blood fat, protecting low density lipoprotein, inhibiting PAF (platelet activating factor), inhibiting thrombosis, scavenging free radicals and the like; the rhizoma Dioscoreae extract contains polysaccharide, starch, protein, free amino acids and microelements, and has effects of regulating immunity, resisting oxidation, lowering blood sugar, reducing blood lipid, resisting tumor, regulating spleen and stomach function, and resisting mutation. Oat beta-glucan has the effects of reducing blood fat and serum cholesterol, and has important effects of preventing and treating cardiovascular and cerebrovascular diseases and diabetes. The galactooligosaccharide is a good nutrition source and effective multiplication factor of beneficial bacteria such as bifidobacterium, lactobacillus acidophilus and the like in human intestinal tracts, and can improve the digestion and absorption functions of the human intestinal tracts.
According to the invention, the prepared probiotic freeze-dried tablet can enhance the effect of probiotics on intestinal microorganism regulation, can assist in reducing blood sugar and blood fat, and has strong effect of improving human immunity by compounding the compound probiotic, the natural plant extract (ginkgo extract and Chinese yam extract), oat beta-glucan, galactooligosaccharide and other main substances.
Further, the probiotic freeze-dried tablet consists of the following components in parts by weight:
30 parts of composite probiotics, 15 parts of galactooligosaccharide, 10 parts of oat beta-glucan, 15 parts of freeze-drying protective agent, 7 parts of ginkgo extract, 3 parts of Chinese yam extract, 1.5 parts of vitamin C, 1.2 parts of vitamin E and 2 parts of stevioside.
Further, the composite probiotic agent prepared by the invention is obtained by culturing lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus according to the weight ratio of 5:3:1:1.
According to the invention, a large number of strains which can promote intestinal tracts in the prior art are screened, and the strains are found that when lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus are used in a combined mode according to the weight ratio of 5:3:1:1, the balance of microbial flora in the intestinal tracts can be maintained by inhibiting the growth and reproduction of pathogenic bacteria; promote the absorption of nutrient substances by human body and has a certain immunoregulation effect on human body; the method can reduce the content of serum cholesterol in a human body, prevent cardiovascular diseases and the like, and meanwhile, the added probiotics of different types are mixed and cultured, so that the synergistic effect among the probiotics can be fully exerted, the viable count of the probiotics is increased, and the method has important influence on improving the stability of the probiotic preparation in a storage period and better exerting the regulating effect on human intestinal tracts.
It is known from the prior art that only if the complex probiotics are colonized in the human intestinal tract in a living state and in sufficient quantity, nutrients beneficial to the human body can be produced through normal physiological metabolism. However, exposure of living cells to additional stress pressure during lyophilization tends to cause loss of cell activity, osmotic shock, formation of intracellular ice crystals and recrystallization can also cause cell membrane damage, resulting in partial living cell death. Therefore, a freeze-drying protective agent needs to be added in the freeze-drying process to protect cells and avoid the phenomenon that the survival rate of viable bacteria in the freeze-dried powder is reduced due to cell damage.
The freeze-drying protective agent added in the invention comprises the following components in parts by weight:
10-16 parts of sodium starch octenyl succinate, 6-10 parts of pullulan polysaccharide, 3-9 parts of low-substituted hydroxypropyl cellulose, 5-8 parts of 6-hydroxy kaempferol, 1-5 parts of arginine, 1-3 parts of L-cysteine, 1-3 parts of propylene glycol alginate, 1-3 parts of calcium carbonate and 30-60 parts of deionized water.
Further, the freeze-drying protective agent consists of the following components in parts by weight:
12 parts of sodium starch octenyl succinate, 8 parts of pullulan, 6 parts of low-substituted hydroxypropyl cellulose, 6 parts of 6-hydroxy kaempferol, 3 parts of arginine, 2 parts of L-cysteine, 2 parts of propylene glycol alginate, 1.5 parts of calcium carbonate and 50 parts of deionized water.
Because different protective agents have different action mechanisms on cell protection, lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus are adopted as the compound probiotic, in order to improve the activity of the strain of the compound probiotic after freeze-drying, a great amount of screening and research are carried out on the components of the freeze-drying protective agent, and a plurality of different types of protective agents are adopted for compounding and use, so that the synergistic effect is achieved, and according to experimental results, after the freeze-drying protective agent is added, the survival rate of the strain of the compound probiotic after vacuum freeze-drying can be effectively improved, and the survival rate of the strain is more than 92%; the storage stability of the probiotics freeze-dried tablet prepared by the method can be effectively improved, and the problem that the viable count of probiotics is reduced due to environmental problems in the transportation and storage processes is effectively solved; meanwhile, the gastric acidity resistance of the probiotic freeze-dried tablet can be improved, and the activity of the probiotic freeze-dried tablet in the gastric digestion process is protected from being damaged, so that probiotics can be planted in intestinal tracts, and the health-care effect of the probiotics is better exerted.
Further, the preparation method of the lyoprotectant comprises the following steps:
dissolving starch sodium octenyl succinate, pullulan polysaccharide, low-substituted hydroxypropyl cellulose, 6-hydroxy kaempferol, arginine, L-cysteine, propylene glycol alginate and calcium carbonate in deionized water, and stirring uniformly to obtain the final product.
The invention also provides a preparation method of the probiotic freeze-dried tablet, which comprises the following steps:
s1, inoculating the frozen and preserved strains into an MRS liquid culture medium respectively, and subculturing for 1-3 times to obtain activated strains;
s2, mixing the activated strains obtained in the step S1 according to a proportion, inoculating the mixed strains into an MRS liquid culture medium for culture, centrifuging the cultured culture solution, removing supernatant to retain bacterial mud, and washing the bacterial mud with inactivated physiological saline to obtain the composite probiotic;
and S3, adding a freeze-drying protective agent into the composite probiotic agent obtained in the step S2, stirring uniformly, adding galactooligosaccharide, oat beta-glucan, ginkgo extract, chinese yam extract, vitamin C, vitamin E and stevioside, stirring uniformly, putting into a vacuum freeze dryer for freeze drying, and tabletting.
Further, in the step S1, the culture temperature is 35-38 ℃, and the culture time is 20-28 h.
Further, the total inoculation amount in the step S2 is 1.5-2.5%, the culture temperature is 37 ℃, and the culture time is 18-24 hours;
the centrifugal rotating speed in the step S2 is 4000-8000 rpm, and the centrifugal time is 10-15 min; the bacterial mud is washed for 1-2 times by using the inactivated normal saline.
Further, the specific step of freeze-drying in the step S3 is as follows: and (3) placing the materials into a vacuum freeze dryer, pre-freezing for 2-3 hours at the temperature of-80 to-70 ℃, vacuumizing to the vacuum degree of 10-20 pa, heating to the temperature of-50 to-40 ℃, and freeze-drying for 24-28 hours.
Compared with the prior art, the probiotics freeze-dried tablet and the preparation method thereof provided by the invention have the following advantages:
(1) According to the invention, through screening different types of probiotics for mixed culture, the synergistic effect among the probiotics can be fully exerted, and the prepared probiotic freeze-dried tablet can effectively maintain the balance of microbial flora in intestinal tracts, promote the absorption of nutrient substances by human bodies and have a certain immunoregulation effect on human bodies.
(2) According to the probiotic freeze-dried tablet provided by the invention, through reasonably compounding different types of freeze-drying protective agents, the prepared probiotic freeze-dried tablet has excellent freeze-drying resistance and storage stability, can more effectively resist inactivation or death of probiotics caused by a freeze-drying process in a freeze-drying process, improves the survival rate of live bacteria in the prepared freeze-dried tablet, and can also improve the gastric acidity resistance of the probiotic freeze-dried tablet, so that the probiotics can be planted in intestinal tracts, and the health-care effect of the probiotics can be better exerted.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention as long as they do not depart from the basic idea of the invention.
In the following examples and comparative examples, the reagents not specifically described were conventional reagents, and were purchased from conventional reagent manufacturers and sales companies, and the information of some raw material manufacturers and the like was as follows:
the Dioscorea opposita extract was purchased from Nanjing daosf biotechnology limited;
6-hydroxy kaempferol, CAS no: 4324-55-4, available from Shanghai Seiyaka Biotechnology Co., ltd;
safflower extract, CAS number: 36338-96-2; ginkgo extract, CAS no: 90045-36-6 from Shaanxi North Biotechnology Co.
Example 1A probiotic freeze-dried tablet and its preparation method
A probiotic freeze-dried tablet consists of the following components in parts by weight:
20 parts of composite probiotics, 10 parts of galactooligosaccharide, 8 parts of oat beta-glucan, 10 parts of freeze-drying protective agent, 5 parts of ginkgo extract, 2 parts of Chinese yam extract, 0.5 part of vitamin C, 0.8 part of vitamin E and 1.2 parts of stevioside.
The freeze-drying protective agent consists of the following components in parts by weight:
10 parts of sodium starch octenyl succinate, 6 parts of pullulan, 3 parts of low-substituted hydroxypropyl cellulose, 5 parts of 6-hydroxy kaempferol, 1 part of arginine, 1 part of L-cysteine, 1 part of propylene glycol alginate, 1 part of calcium carbonate and 30 parts of deionized water.
The preparation method of the freeze-drying protective agent comprises the following steps:
dissolving starch sodium octenyl succinate, pullulan polysaccharide, low-substituted hydroxypropyl cellulose, 6-hydroxy kaempferol, arginine, L-cysteine, propylene glycol alginate and calcium carbonate in deionized water, and stirring uniformly to obtain the final product.
The preparation method of the probiotic freeze-dried tablet comprises the following steps:
s1, inoculating cryopreserved strains (lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus) into an MRS liquid culture medium respectively, culturing at 35 ℃ for 28 hours, and subculturing for 2 times to obtain activated strains;
s2, taking the activated strain obtained in the step S1, mixing lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus according to the weight ratio of 5:3:1:1, inoculating the mixture into an MRS liquid culture medium, culturing the mixture at 37 ℃ for 18 hours, centrifuging the cultured culture solution at 4000rpm for 15 minutes, removing supernatant to retain bacterial mud, and washing the bacterial mud for 1 time by using inactivated physiological saline to obtain the composite probiotic;
and S3, adding a freeze-drying protective agent into the composite probiotic agent obtained in the step S2, stirring uniformly, adding galactooligosaccharide, oat beta-glucan, ginkgo extract, chinese yam extract, vitamin C, vitamin E and stevioside, stirring uniformly, putting into a vacuum freeze dryer for freeze drying, and tabletting.
The freeze drying comprises the following specific steps: and (3) placing the materials into a vacuum freeze dryer, pre-freezing for 2 hours at the temperature of-80 ℃, vacuumizing to the vacuum degree of 10pa, heating to the temperature of-50 ℃, and freeze-drying for 24 hours to obtain the finished product.
Example 2A probiotic freeze-dried tablet and its preparation method
A probiotic freeze-dried tablet consists of the following components in parts by weight:
30 parts of composite probiotics, 15 parts of galactooligosaccharide, 10 parts of oat beta-glucan, 15 parts of freeze-drying protective agent, 7 parts of ginkgo extract, 3 parts of Chinese yam extract, 1.5 parts of vitamin C, 1.2 parts of vitamin E and 2 parts of stevioside.
The freeze-drying protective agent consists of the following components in parts by weight:
12 parts of sodium starch octenyl succinate, 8 parts of pullulan, 6 parts of low-substituted hydroxypropyl cellulose, 6 parts of 6-hydroxy kaempferol, 3 parts of arginine, 2 parts of L-cysteine, 2 parts of propylene glycol alginate, 1.5 parts of calcium carbonate and 50 parts of deionized water.
The preparation method of the freeze-drying protective agent comprises the following steps:
adding starch sodium octenyl succinate, pullulan polysaccharide, low-substituted hydroxypropyl cellulose, 6-hydroxy kaempferol, arginine, L-cysteine, propylene glycol alginate and calcium carbonate into deionized water, and stirring uniformly to obtain the final product.
The preparation method of the probiotic freeze-dried tablet comprises the following steps:
s1, inoculating cryopreserved strains (lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus) into an MRS liquid culture medium respectively, culturing for 24 hours at 37 ℃, and subculturing for 2 times to obtain activated strains;
s2, taking the activated strain obtained in the step S1, mixing lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus according to the weight ratio of 5:3:1:1, inoculating the mixture into an MRS liquid culture medium, culturing the mixture at the total inoculum size of 2.0 percent at 37 ℃ for 22 hours, centrifuging the cultured culture solution at 6000rpm for 12 minutes, removing supernatant to retain bacterial mud, and washing the bacterial mud for 2 times by using inactivated physiological saline to obtain the composite probiotic;
and S3, adding a freeze-drying protective agent into the composite probiotic agent obtained in the step S2, stirring uniformly, adding galactooligosaccharide, oat beta-glucan, ginkgo extract, chinese yam extract, vitamin C, vitamin E and stevioside, stirring uniformly, putting into a vacuum freeze dryer for freeze drying, and tabletting.
The freeze drying comprises the following specific steps: the materials are put into a vacuum freeze dryer for pre-freezing for 3 hours at the temperature of minus 75 ℃, then vacuumized, the vacuum degree is 15pa, the temperature is raised to minus 45 ℃, and the materials are freeze-dried for 26 hours.
Example 3A probiotic freeze-dried tablet and method for preparing the same
A probiotic freeze-dried tablet consists of the following components in parts by weight:
40 parts of composite probiotics, 20 parts of galactooligosaccharide, 12 parts of oat beta-glucan, 20 parts of freeze-drying protective agent, 8 parts of ginkgo extract, 5 parts of Chinese yam extract, 2 parts of vitamin C, 1.8 parts of vitamin E and 2.5 parts of stevioside.
The freeze-drying protective agent consists of the following components in parts by weight:
16 parts of sodium starch octenyl succinate, 10 parts of pullulan, 9 parts of low-substituted hydroxypropyl cellulose, 8 parts of 6-hydroxy kaempferol, 5 parts of arginine, 3 parts of L-cysteine, 3 parts of propylene glycol alginate, 3 parts of calcium carbonate and 60 parts of deionized water.
The preparation method of the freeze-drying protective agent comprises the following steps:
adding starch sodium octenyl succinate, pullulan polysaccharide, low-substituted hydroxypropyl cellulose, 6-hydroxy kaempferol, arginine, L-cysteine, propylene glycol alginate and calcium carbonate into deionized water, and stirring uniformly to obtain the final product.
The preparation method of the probiotic freeze-dried tablet comprises the following steps:
s1, inoculating cryopreserved strains (lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus) into an MRS liquid culture medium respectively, culturing for 20 hours at 38 ℃, and subculturing for 3 times to obtain activated strains;
s2, taking the activated strain obtained in the step S1, mixing lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus according to the weight ratio of 5:3:1:1, inoculating the mixture into an MRS liquid culture medium, culturing the mixture at 37 ℃ for 24 hours, centrifuging the cultured culture solution at 8000rpm for 10min, removing supernatant to retain bacterial mud, and washing the bacterial mud for 2 times by using inactivated physiological saline to obtain the composite probiotic;
and S3, adding a freeze-drying protective agent into the composite probiotic agent obtained in the step S2, stirring uniformly, adding galactooligosaccharide, oat beta-glucan, ginkgo extract, chinese yam extract, vitamin C, vitamin E and stevioside, stirring uniformly, putting into a vacuum freeze dryer for freeze drying, and tabletting.
The freeze drying comprises the following specific steps: the materials are put into a vacuum freeze dryer for pre-freezing for 3 hours at the temperature of-70 ℃, then the vacuum is pumped, the vacuum degree is 20pa, the temperature is raised to-40 ℃, and the freeze drying is carried out for 28 hours.
Comparative example 1A probiotic freeze-dried tablet and a method for preparing the same
In comparison with example 2, comparative example 1 was different in that sodium starch octenyl succinate in the lyoprotectant was replaced with acacia, and the other components were the same as in example 2.
Comparative example 2A probiotic freeze-dried tablet and a method for preparing the same
In comparison with example 2, the difference of comparative example 2 is that pullulan was not added to the lyoprotectant, the amount of sodium starch octenyl succinate was increased to 20 parts, and the other components were the same as in example 2.
Comparative example 3A probiotic freeze-dried tablet and a method for preparing the same
Comparative example 3 is different from example 2 in that 6-hydroxykaempferol in the lyoprotectant is replaced with safflower extract, and other components are the same as those of example 2.
Comparative example 4A probiotic freeze-dried tablet and a method for preparing the same
Comparative example 4 was different from example 2 in that 6-hydroxykaempferol was not added to the lyoprotectant, the amount of L-cysteine was increased to 8 parts, and the other components were the same as in example 2.
Test example one, the survival rate of the probiotic freeze-dried tablet of the invention is detected
1. Test materials: the probiotics freeze-dried tablets prepared in the embodiment 1-3 and the comparative embodiment 1-4 are prepared.
2. The test method comprises the following steps:
(1) Strain survival rate detection: the probiotic quantity changes before and after freezing in examples 1-3 and comparative examples 1-4 of the present invention were respectively measured, viable count was performed by plate counting, and the survival rate was calculated as the ratio of the total number of bacteria after freeze-drying to the total number of bacteria before freeze-drying, wherein: the method for detecting the total bacteria before freeze drying comprises the following steps: in the preparation processes of examples 1 to 3 and comparative examples 1 to 4, detection is performed after the end of step S2, viable bacteria count is performed by adopting a plate counting method, and the result is the total number of bacteria before freeze drying, and the detection method of the total number of bacteria after freeze drying is as follows: in the preparation processes of examples 1 to 3 and comparative examples 1 to 4, detection was performed after the end of step S3, and viable bacteria were counted by a plate count method, and the result was the total number of bacteria after freeze-drying. The results of the survival rate of the strain after freeze-drying are shown in Table 1.
(2) And (3) detecting storage stability of the probiotics freeze-dried tablets: the probiotic freeze-dried tablets prepared in the embodiment 1 to 3 and the comparative example 1 to 4 of the invention are stored in a sealed manner for 6 months under the conditions that the temperature is 25+/-2 ℃ and the relative humidity is 40+/-5%, then viable bacteria are counted by adopting a plate counting method, the result is the total number of bacteria stored for 6 months, the survival rate is calculated, and the survival rate is the ratio of the total number of bacteria stored for 6 months after freeze drying to the total number of bacteria before freeze drying. The results of the survival rate of the strain after 6 months of standing are shown in Table 1.
3. Test results:
the strain survival rate and the storage stability detection result of the probiotic freeze-dried tablet prepared by the invention are shown in table 1.
TABLE 1
As can be seen from Table 1, the survival rate of the strain after vacuum freeze-drying of the probiotic freeze-dried tablets prepared in the embodiments 1-3 of the present invention is still more than 92%, and the survival rate of the strain after 6 months of standing still can reach more than 83%, wherein the best effect of the embodiment 2 is the best embodiment of the present invention. And when the components of the freeze-drying protective agent are changed in comparative examples 1-4, the effect is obviously reduced compared with the survival rate of the example 2, which shows that the components of the freeze-drying protective agent provided by the invention have obvious synergistic effect, can effectively avoid cell damage caused by vacuum freeze drying of cells, and simultaneously improve the storage stability of the probiotic freeze-dried tablet, and the survival rate of strains is not greatly influenced after the probiotic freeze-dried tablet is placed for 6 months.
Test example two, artificial gastric juice tolerance test
1. Test materials: the probiotics freeze-dried tablets prepared in the embodiment 1-3 and the comparative embodiment 1-4 are prepared.
2. The test method comprises the following steps: firstly, detecting the viable count of the probiotic freeze-dried tablets prepared in the embodiment 1-3 and the comparative example 1-4 (before being placed in artificial gastric juice), then respectively taking 1g of the probiotic freeze-dried tablets prepared in the embodiment 1-3 and the comparative example 1-4, placing the probiotic freeze-dried tablets in a triangular flask with 50mL of preheated artificial gastric juice (pH1.2), uniformly mixing, vibrating in a constant temperature bed (180 r/min, 37+/-1 ℃), sampling at the 30 th, 60 th and 90 th minutes respectively, measuring the viable count by adopting a plate counting method, and taking an average value by each group of parallel three tests, thereby determining the survival rate of the thalli at the moment. The survival rate of the probiotics is the ratio of the number of live bacteria when sampling is carried out for 30, 60 and 90 minutes respectively to the number of live bacteria of the probiotics before the artificial gastric juice is placed.
The preparation method of the artificial gastric juice comprises the following steps: weighing 10 g pepsin, measuring 16.4 mL hydrochloric acid (0.1 mol/L) by a measuring cylinder, adding water into a beaker, stirring uniformly, filtering, fixing the volume to a 1000 mL volumetric flask, and adjusting the pH of the artificial gastric juice to 1.2,0.2 mu m by a pH meter, filtering and sterilizing by a sterile microporous filter membrane for later use.
3. Test results
The tolerance test results of the probiotic freeze-dried tablet prepared by the invention in artificial gastric juice are shown in table 2.
TABLE 2
As can be seen from Table 2, in the case of gastric acid simulation, the survival rate of the probiotics in the freeze-dried tablet prepared in examples 1 to 3 of the present invention can still reach 82.9% or more after 90min in gastric juice simulation, and the survival rate of the probiotics in gastric juice simulation is reduced to different degrees when the composition of the freeze-drying protective agent is changed in comparative examples 1 to 4. The invention shows that after the freeze-drying protective agent provided by the invention is added when the vacuum freeze-drying protective agent is adopted, the acid resistance of probiotics in gastric juice can be effectively improved, and the activity of the probiotics in the gastric digestion process is protected from being damaged.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (8)
1. The probiotics freeze-dried tablet is characterized by comprising the following components in parts by weight:
20-40 parts of a composite probiotic, 10-20 parts of galactooligosaccharide, 8-12 parts of oat beta-glucan, 10-20 parts of a freeze-drying protective agent, 5-8 parts of a gingko extract, 2-5 parts of a Chinese yam extract, 0.5-2 parts of vitamin C, 0.8-1.8 parts of vitamin E and 1.2-2.5 parts of stevioside; the compound probiotic is prepared by inoculating and mixing lactobacillus rhamnosus, lactobacillus acidophilus, bifidobacterium breve and lactobacillus pentosus according to the weight ratio of 5:3:1:1, and culturing; the freeze-drying protective agent consists of the following components in parts by weight: 10-16 parts of sodium starch octenyl succinate, 6-10 parts of pullulan polysaccharide, 3-9 parts of low-substituted hydroxypropyl cellulose, 5-8 parts of 6-hydroxy kaempferol, 1-5 parts of arginine, 1-3 parts of L-cysteine, 1-3 parts of propylene glycol alginate, 1-3 parts of calcium carbonate and 30-60 parts of deionized water.
2. The probiotic freeze-dried tablet according to claim 1, which is composed of the following components in parts by weight: 30 parts of composite probiotics, 15 parts of galactooligosaccharide, 10 parts of oat beta-glucan, 15 parts of freeze-drying protective agent, 7 parts of ginkgo extract, 3 parts of Chinese yam extract, 1.5 parts of vitamin C, 1.2 parts of vitamin E and 2 parts of stevioside.
3. The probiotic freeze-dried tablet according to claim 1, wherein the freeze-dried protective agent consists of the following components in parts by weight: 12 parts of sodium starch octenyl succinate, 8 parts of pullulan, 6 parts of low-substituted hydroxypropyl cellulose, 6 parts of 6-hydroxy kaempferol, 3 parts of arginine, 2 parts of L-cysteine, 2 parts of propylene glycol alginate, 1.5 parts of calcium carbonate and 50 parts of deionized water.
4. The probiotic freeze-dried tablet according to claim 1, wherein the preparation method of the freeze-dried protectant comprises the following steps: dissolving starch sodium octenyl succinate, pullulan polysaccharide, low-substituted hydroxypropyl cellulose, 6-hydroxy kaempferol, arginine, L-cysteine, propylene glycol alginate and calcium carbonate in deionized water, and stirring uniformly to obtain the final product.
5. A method for preparing the probiotic freeze-dried tablet according to any one of claims 1 to 4, comprising the following preparation steps:
s1, inoculating the frozen and preserved strains into an MRS liquid culture medium respectively, and subculturing for 1-3 times to obtain activated strains;
s2, mixing the activated strains obtained in the step S1 according to a proportion, inoculating the mixed strains into an MRS liquid culture medium for culture, centrifuging the cultured culture solution, removing supernatant to retain bacterial mud, and washing the bacterial mud with inactivated physiological saline to obtain the composite probiotic;
and S3, adding a freeze-drying protective agent into the composite probiotic agent obtained in the step S2, stirring uniformly, adding galactooligosaccharide, oat beta-glucan, ginkgo extract, chinese yam extract, vitamin C, vitamin E and stevioside, stirring uniformly, putting into a vacuum freeze dryer for freeze drying, and tabletting.
6. The method for preparing a probiotic freeze-dried tablet according to claim 5, wherein the culturing temperature in the step S1 is 35-38 ℃ and the culturing time is 20-28 h.
7. The method for preparing a probiotic freeze-dried tablet according to claim 5, wherein the total inoculation amount in the step S2 is 1.5-2.5%, the culture temperature is 37 ℃, and the culture time is 18-24 hours; the centrifugal rotation speed is 4000-8000 rpm, and the centrifugal time is 10-15 min; the bacterial mud is washed for 1-2 times by using the inactivated normal saline.
8. The method for preparing a probiotic freeze-dried tablet according to claim 5, wherein the specific step of freeze-drying in step S3 is: and (3) placing the materials into a vacuum freeze dryer, pre-freezing for 2-3 hours at the temperature of-80 to-70 ℃, vacuumizing to the vacuum degree of 10-20 pa, heating to the temperature of-50 to-40 ℃, and freeze-drying for 24-28 hours.
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Effective date of registration: 20240311 Address after: 236000, Room 315, 2.5 Industrial Park, Bowu Modern Industrial Park, Hehuan Road, High tech Zone, Bozhou City, Anhui Province Patentee after: Bafeiba Food Technology Co.,Ltd. Country or region after: China Address before: Room 409, 4th Floor, No. 3, Yiheng Road, Datian, Hebian, Hebian, Helong Street, Baiyun District, Guangzhou City, Guangdong Province, 510000 Patentee before: Guangzhou Bafeiba Health Industry Co.,Ltd. Country or region before: China |